We propose to use the technique of cryoenzymology, i.e., subzero temperatures and fluid aqueous organic solvent systems, to study the mechanisms of catalysis of the following enzymes: ribonuclease A, lysozyme, beta-glucosidase, beta-galactosidase, D-amino acid oxidase, and glucose oxidase. The method has been shown to be well-suited for the detection and accumulation of intermediates in the enzyme-catalyzed reactions of specific substrates (both in the dissolved and crystalline states). By using appropriate spectral methods (e.g., fluorescence, Raman, e.s.r, c.d.) to monitor the reaction after initiation by mixing enzyme and substrate at a very low temperature, e.g., -100 degrees C, it will be possible to obtain kinetic, thermodynamic and some structural information about intermediates and their transformations. Further structural information will be obtained when possible from X-ray diffraction studies on the crystalline trapped intermediates. From the information obtained in this investigation we expect to be able to significantly contribute not only to the understanding of the mechanisms of action of these particular enzymes but also to further knowledge about the factors responsible for the efficiency of enzymes, and the events occurring during the dynamic processes of catalysis in general. The low-temperature technique will also be used to detect and study intermediates in the folding and unfolding of ribonuclease.